Many entities are known for having an affinity for certain inorganic substances. This interaction has been exploited primarily for the purification of appropriately appended recombinant proteins from various growth and other media and a variety of such media and purification protocols are commercially available. For example, a variety of peptidyl sequences are know to have an affinity for inorganic surfaces. Histidine terminated peptide sequences, in particular poly-histidine sequences, are known to have an affinity for certain nanoparticles, such as CdSe/ZnS nanoparticles and quantum dots (QDs). Poly-cysteine residues have been introduced into proteins and peptides recombinantly to facilitate their binding to gold and other nanoparticles and surfaces. Several unique peptide sequences have been selected for their binding to various metal and/or semiconductor surfaces using phage display and other selection/molecular evolution techniques. As non-limiting examples, Table I below illustrates a variety of peptide sequences and the particular inorganic materials for which they are know to have an affinity.
TABLE IInorganic material for whichPeptidyl sequencesthere is an affinityMHGKTQATSGTIQS (SEQ ID NO 1)GoldSKTSLGQSGASLQGSEKLTNG (SEQ ID NO 2)QATSEKLVRGMEGASLHPAKT (SEQ ID NO 3)DRTSTWR (SEQ ID NO 4)PlatinumQSVTSTK (SEQ ID NO 5)SSSHLNK (SEQ ID NO 6)SVTQNKY (SEQ ID NO 7)PalladiumSPHPGPY (SEQ ID NO 8)HAPTPML (SEQ ID NO 9)AYSSGAPPMPPF (SEQ ID NO 10)SilverNPSSLFRYLPSD (SEQ ID NO 11)SLATQPPRTPPV (SEQ ID NO 12)MSPHPHPRHHHT (SEQ ID NO 13)Silicon oxideRGRRRRLSCRLL (SEQ ID NO 14)KPSHHHHHTGAN (SEQ ID NO 15)VKTQATSREEPPRLPSKHRPG (SEQ ID NO 16)ZeolitesMDHGKYRQKQATPG (SEQ ID NO 17)NTRMTARQHRSANHKSTQRA (SEQ ID NO 18)Zinc oxideYDSRSMRPH (SEQ ID NO 19)HTQNMRMYEPWF (SEQ ID NO 20)Calcium carbonateDVFSSFNLKHMR (SEQ ID NO 21)VVRPKAATN (SEQ ID NO 22)Chromium oxideRIRHRLVGQ (SEQ ID NO 23)RRTVKHHVN (SEQ ID NO 24)Iron oxideAQNPSDNNTHTH (SEQ ID NO 25)Gallium arsenideRLELAIPLQGSG (SEQ ID NO 26)TPPRPIQYNHTS (SEQ ID NO 27)NNPMHQN (SEQ ID NO 28)Zinc sulfideSee M. Sarikaya, et al. “Molecular biomimetics: nanotechnology through biology,” Nature Materials, Vol. 2, pp. 577-585 (September 2003), which is incorporated herein by reference in its entirety.
Nanocrystals and QDs are generally composed of metals, metal oxides and semiconductors, all substantially inorganic materials. Nanoparticles and QDs display unique spectroscopic and electronic properties distinct from molecular compounds or parent bulk materials. QDs have been widely demonstrated as useful tools and probes for the development of highly sensitive biological and other types of multiplexing assays, i.e. the simultaneous detection of multiple signals. The substantially inorganic nature of QDs and nanoparticles makes it difficult to conjugate them to organic substances by standard methodologies. As such, there are only limited methods available for coupling nanoparticles and QDs to organic substances, such as proteins or peptides (or any biomolecule), many of which result in a heterogeneous composite structures or aggregates. These concerns continue to hinder progress in this field.
Current methods for linking organic substances to QDs involve multiple steps, are cumbersome and not practical. As a result, current methods for linking organic substances and inorganic substances are only suitable for very specific conjugation applications. While specific chemistries may have been developed to join a particular organic substance to a particular inorganic substance, the process for doing so is often very complex, can take several days to proceed and may not be applicable to other organic or inorganic substances pairs. For example, biomolecule attachment to a functionalized QD is usually achieved by employing a large excess of the biomolecule, frequently resulting in a QD to biomolecule ratio that cannot be controlled, cross reactivity, aggregation and precipitation. As such, the aggregated size of the final QD-biomolecule conjugate may be too large so as to preclude certain applications. Further, the chemistries and methods used to link inorganic substances to organic substances are generally unstable reactions, requiring the immediate conjugation of the organic substance and/or inorganic substance without degrading of the intermediate linker. Further, the actual conjugation steps often require several subsequent purification steps, making the process of conjugation selective to the particular protein, complex and burdensome.